Using cells reprogrammed with oncogenic factors for screening anti-neoplastic agents

ABSTRACT

Cells reprogrammed with oncogenic factors (CROFs) include incorrectly programmed stem cells such as induced pluripotent stem cells (iPSCs). The present application discloses linkages between iPS reprogramming and its potential roles in neoplastic transformation and thus establishes a foundation for using iPSCs as a class of CROFs for screening anti-neoplastic agents. The screening process involves examining the capability of a single agent or a combination of multiple agents in suppressing a neoplastic process including aerobic glycolysis and the related anabolism and thus inhibiting excessive reproduction of the neoplastic cell. In addition, agents are also screened for their potential in inhibiting invasion and migration of neoplastic cells. Anti-neoplastic agents found through methods disclosed here may represent wide-spectrum anti-neoplastic agents but possess very limited side effect because they target at one or more unique aspects of neoplastic processes, rather than affecting one or more living processes common to all cells including normal cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

Not Applicable.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable.

TECHNICAL FIELD

The invention involves methods for screening agents effective ininhibiting neoplastic aerobic glycolysis and anabolism supportingexcessive cell reproduction and in reducing invasion and migration ofneoplastic cells. More specifically, the invention utilizes cellsreprogrammed with oncogenic factors (CROFs) including inducedpluripotent stem cells (iPSCs) as means for screening agents thatspecifically inhibit the neoplastic aerobic glycolysis and anabolism aswell as the invasive and metastatic properties of neoplastic cells.

BACKGROUND OF THE INVENTION

Neoplasia is the abnormal proliferation of cells which means thereproduction of these cells exceeds, and is uncoordinated with, that ofthe normal tissues around them.

Neoplasm is an abnormal mass of tissue resulted from neoplasia. It mayappear as tumor or cancer. Tumor represents a localized swelling formedby a solid neoplasm. Cancer represents a disease in which the neoplasticcells displayed not only uncontrolled growth (reproduction) but alsoinvasion (intrusion on and destruction of adjacent tissues) andsometimes metastasis (dispersion to other locations in the body vialymph or blood). Neoplasm can be benign or malignant. Benign neoplasmoften contains highly differentiated cells. Malignant neoplasm such ascancer often contains poorly differentiated or undifferentiated cells.

Despite great progresses in understanding its etiology and pathologyneoplasia, especially the malignant neoplasia, still remains as a greatrisk to human life. This is because there is still a lack of effectivetreatment for neoplasm, especially the malignant neoplasm. Manyconventional anti-neoplastic agents suffer from drawbacks of severe sideeffects due to their inhibition on some common processes shared bynormal cells [1-3]. On the other hand, highly-specific anti-neoplasmdrugs that target at the specific “leaf” level genetic mutation uniqueto different neoplastic cells are not only too narrow spectrum in theiranti-neoplastic effect but also suffer from losing effect as survivingneoplastic cells can develop resistance by using alternative routes[4-5]. More ironically, some anti-cancer agents can kill the targetedoriginal cancer cells but meantime lead to the formation of new cancerin the patients [6-8]. The sad reality is that most anticancer drugs[9-10] really do not offer much meaningful benefit to patients' qualityof life because they add just an extra week or two suffering time to thepatients' lifespan [11-12].

Thus, an urgent need in combating neoplasia is to obtain some “rootkillers” for neoplastic cells [13]. In other words, we need to findwide-spectrum anti-neoplastic agents that are harmful only to neoplasticcells.

In order to find such a “root” killer of neoplasia, we need tounderstand the “root” of neoplasia or the “Achilles' heel” of neoplasm.

The “root” of neoplasia or the “Achilles' heel” of neoplasm resides inthe unique metabolic properties and the living processes of theneoplastic cells.

A hallmark of all neoplasms is their high rate of glycolysis even undera high oxygen concentration. This phenomenon has been known as Warburgeffect since 1930s but remains poorly understood even today [14]. Duringaerobic glycolysis, pyruvate generated from glucose is not transportedinto mitochondria for total oxidation for yielding more energy but isconverted to lactate in cytosol and then excreted outside the cell [15].For long time, it is unknown why neoplastic cells would “waste” glucoseand choose an energy-“inefficient” metabolism. However, the finding of a“neoplastic or pathological Cori cycle” in which the excreted lactate iscarried by the blood to the liver and converted to glucose for reuse bythe neoplasm may shed some light in understanding Cachexia, a conditionexists in neoplastic patients who suffer massive loss of normal bodymass as the neoplasm continues its growth [16]. It turns out that, byavoiding a complete “burn” of glucose to CO₂, neoplastic cells preservedsome key carbon “skeleton” for anabolism. The combined result of Warburgeffect (the aerobic glycolysis) and pathological Cori cycle (theneoplastic anabolism) thus causes a metabolic imbalance: shiftingresource toward neoplastic cells and away from normal cells. Thismetabolic imbalance ultimately results in systematic failure of patientssuffer from a neoplastic disease and their death.

In addition, recent studies have shown that some of the molecularmechanisms underlying the neoplastic metabolism also influence invasionand migration of malignant neoplastic cells which are responsible formetastasis and a wider range of neoplastic diseases [17].

Thus, a very effective and much needed approach for treating neoplasiashould be based on inhibiting aerobic glycolysis (Warburg effect),neoplastic anabolism (pathological Cori cycle), or both at the sametime. This approach should yield therapeutic schemes that present muchless side effects than those associated with conventional anti-cellcycle (reproduction)-based therapy such as chemotherapy or radiotherapy.This approach should also yield much broader spectrum anti-neoplasticdrugs than those approaches targeting at some rare mutations specificfor only a certain type of neoplastic cells. Understanding uniquemetabolism common to all or at least most neoplastic cells and findingagents inhibiting such neoplastic metabolism may hit the Achilles' heelof neoplasm and eradicate neoplasm from its root.

In addition to the still lacking full translation of the existinginsights into the causes of cancer from bench to bed, a major obstaclein searching for anti-neoplastic agents is ironically the shortage ofneoplastic cells suitable for laboratory drug screening [18]. Despitethe high incidence of neoplasia, neoplastic cells preserved for researchuse are relatively few. Collecting primary neoplastic cells is a complexprocedure impeded with many red tapes, not to say the great investmentsand efforts required for characterizing them for research use.Consequently, only a limited number of primary cancer cells have beenestablished as useful cell lines. Thus, there is a need to find moremodel neoplastic cells for utilization in screening anti-neoplasticagents.

In addition to this limitation in quantity, some neoplastic cell linespassed many times in the laboratories might have accumulated additionalmutations that are atypical for natural neoplasm [18]. Thus, it is notuncommon that drugs effective against laboratory lines of cancer cellsactually failed dramatically in clinical trails. [19]. Some of theanti-cancer drugs actually lead to opposite effects [20-21]. It is alsoknown that some conventional chemotherapy sometimes induces tumorregression while simultaneously elicits stress responses that protectsubsets of tumor cells [22]. Thus, there is a need for finding moreappropriate model neoplastic cells suitable for reliable screening ofanti-neoplastic agents.

BRIEF SUMMARY OF THE INVENTION

It is an object of the present invention to identify cells reprogrammedwith oncogenic factors (CROFs) as surrogate neoplastic cells forscreening anti-neoplastic agents.

It is another object of the present invention to classify inducedpluripotent stem cells (iPSCs) as a group of CROFs suitable forscreening anti-neoplastic agents.

It is an object of the present invention to provide methods forscreening agents, in single or in combination, against neoplastic cellsby detecting its or their capability of inhibiting aerobic glycolysis.

It is another object of the present invention to provide methods forscreening agents, in single or in combination, against neoplastic cellsby detecting its or their capability of inhibiting neoplastic anabolism.

It is an object of the present invention to provide methods forscreening agents, in single or in combination, against neoplastic cellsby detecting its or their capability of inhibiting invasion, migrationand metastasis.

DETAILED DESCRIPTION OF THE INVENTION

Recently, cells known as induced pluripotent stem cells (iPSCs) havebeen generated in large quantity and diversity [23-25]. These iPSCs havebeen described as “indistinguishable” from embryonic stem cells (ESCs)[26-28] and thus been perceived as “ethical” and “safe” replacements ofESCs for cell therapy [29-31] and even regenerative medicine [32-35].

It is a discovery of the present inventor that iPSCs are incorrectlyprogrammed stem cells (still abbreviated as iPSCs) or, in other words,man-made cancer stem cells (mmCSCs) [36-38]. Thus, iPSCs can beclassified as a group of CROFs, cells reprogrammed with oncogenicfactors, and can be used as replacements for naturally-occurring cancercells in screening agents against neoplastic cells.

It is a further discovery of the present inventor that iPS reprogrammingcan be linked with neoplastic aerobic glycolysis and anabolism [39].Thus, iPSCs can be used as model cells for screening agents thatspecifically inhibit some aerobic glycolysis and anabolismcharacteristic for neoplasia.

In addition, it is a discovery of the present inventor that iPSCs maypossess invasion and migration capability common to malignant neoplasticcells and thus may also be used for screening agents against theinvasion, migration and metastasis of neoplastic cells.

It should be pointed out that the present invention of using iPSCs as aclass of CROFs for screening anti-neoplastic agents is fundamentallydifferent from those inventions of using iPSCs as non-cancerous cellsfor modeling other diseases [40-41] and screening drugs against thosediseases [42-43]. As a matter of fact, iPS researchers have been focusedon inventing methods for making iPSCs [44] because human iPSCs have beenperceived as “less complicated” “human pluripotent cells” than embryonicstem cells (ESCs) and thus are “potentially useful in therapeuticapplications in regenerative medicine” [44]. More significantly, claimsof generating “cancer-free” “safe” iPSCs suitable for clinicalapplications are being made repeatedly [28, 45-47], despite thecriticisms against the hype contained in these claims [36, 48]. A veryrecent publication [49] describes acquisition of iPSCs by selectingthose cells with transgenes integrated into the so-called “safeharbors”, the genomic regions outside positions known for integrationmutation. Even though the iPSCs are still made with the already knownoncogenes, a claim of “out of harm's way is still made [50]. Thisdemonstrates the lacking of understanding with regarding to how iPSreprogramming results in neoplastic transformation and thus how similariPSCs are to cancer cells than to ESCs.

At the present time mainstream iPS researchers and top journals arestill rejecting the discovery of iPSCs as man-made cancer stem cells(mmCSCs) [37-38] which identifies some cancer risks for various“cancer-free” iPSCs [23-24, 26, 51-53]. Strong efforts are still beingmade in promoting iPSCs as “ethical” and “safe” ESC replacements forcell therapy and regenerative medicine [28, 54-58]. A recent publicationreporting generating iPSCs from dermal fibroblasts of a patientsuffering from Hutchinson-Gilford Progeria syndrome (HGPS) [59] has evenbeen regarded as to “lead to novel insights into mechanisms ofaging”[60], even though these HGPS-iPSCs are merely some cancerous cellscarrying the mutations for HGPS.

Thus, even though some recent publications have noticed the “similarity”between iPSCs and cancer cells [61-62] or the common path between thegeneration of iPSCs and CSCs [63], the authors of these publications arestill contributing the intriguing “parallel” as a result of partial [28]or incomplete [47] reprogramming. At the end, the intrinsic cancer riskof iPSCs has been neglected even in the “comprehensive review [28] or“straight talk” [47] by the leading iPS researchers. Arguments have alsobeen made that “although there are common pathways activated duringreprogramming and tumorigenesis, there are fundamental differencesbetween iPS and transformed cancer stem cells” [63].

However, by clearly identifying the various linkages between iPSreprogramming and neoplastic transformation, it is hoped that thepresent application will establish a solid foundation for arguing iPSCsas a kind of neoplastic cells very similar to natural cancer cells andthus justifying their use as serendipitous replacements for cancer cellsin screening agents against neoplastic cells.

It turns out that Myc (c-Myc), a very important iPS reprogrammingfactor, is a notorious oncogene and a master transcription factor thatintegrates cell proliferation with metabolism through its regulation ofthousands of genes including microRNAs (miRNA) [64]. In addition to itsknown function in regulating the cell cycle and glucose metabolism [65],Myc also stimulates glutamine catabolism [66] through the repression ofmiRNAs miR-23a and miR-23b [67].

More significantly c-Myc enhances the expression of poly-pyrimidinetract binding protein PTB (also known as hnENPI), hnRNA1 and hnRNA2, andleads to selective expression of pyruvate kinase isoform 2 (PKM2) [65].PKM2 is the M2 splice isoform of pyruvate kinase (PK) [68] which is akey enzyme for aerobic glycolysis [69], as compared with the M1 spliceisoform of pyruvate kinase (PKM1) which is a key enzyme for oxidativephosphorylation. Thus the selective expression of specific isoform of PKserves as a toggle switch for shifting mass-energy metabolism between anenergy production-efficient oxidative phosphorylation and a massproduction-efficient aerobic glycolysis.

It is interesting to notice that PKM2 is the dominant form of PK inembryonic cells and PKM1 is the dominant form of PK in the adult cells[68-69]. This age-specific expression of different isoforms of the sameenzyme reflects the physiological need as PKM2 is needed for glycolyticanabolism supporting mass increase in the growth stage of the life andPKM1 is needed for allowing established cells to perform moreenergy-consuming functions at the grown up stage of the life. Thus achange in the expression of different isoforms of the same enzyme leadsto different modes of metabolism in different life stages. Overgrowth ofcell mass such as neoplasm is not a desired shift.

Unfortunately, the re-expression of PKM2 [70] activates those “resting”cells and drives them from normally a “quiescent” state into ahyper-proliferation state [71]. This adulthood expression of anembryonic enzyme isoform does not lead to the “rejuvenation” of thewhole organism but a formation of some harmful and even deadly neoplasm.

More than just contributing to the transformation of normal cells intoneoplastic cells, c-Myc coordinately regulates the expression of 13different “poor-outcome” cancer signatures [17]. In addition, functionalinactivation of MYC in human breast cancer cells specifically inhibitsdistance metastasis in vivo and invasive behavior in vitro [17]. Soc-Myc may also contribute to the acquisition of metastatic capability ofthe neoplastic cells.

Therefore, iPSCs generated with inducing factors including c-Myc maynaturally possess some basic neoplastic features known to natural cancercells.

The iPSCs generated without c-Myc may also possess oncogenic nature. Forexample, Lin28, an inducing factor used in place of c-Myc [25], hasrecently been found in association with cancers [72-74]. Moreimportantly, Lin28 has been shown as a Myc-downstream factor exertingthe similar effect as Myc [74-75].

As a matter of fact, iPS reprogramming factors currently employed aremore or less associated with various cancers [37-38, 76-77]. Thus, theoncogenic potential is intrinsic for iPS reprogramming, at least for theproto-type iPS reprogramming methods [39]. This intrinsic oncogenicpotential is intensified when a tumor-suppressing mechanism is inhibitedor knocked out [39]. Unfortunately, many iPS researchers just do notwant to face with this dark side of iPS reprogramming and continue atlooking at the “bright” side of their discoveries [78]. They emphasizethe enhanced “efficiency” of iPS reprogramming and elevated yield of theiPSCs by knocking out the tumor-suppressing mechanisms [79-83] whileignore the increased risks of cancer potential from these tumorsuppression mechanism-jeopardized iPSCs [84-85].

Nevertheless, increasing reports are presenting observations ofchromosomal aberrations [86] and cancer-related epigenome changes [87]in iPSCs. There are also reports documenting formation ofrhabdomyosarcomas iPSCs [88]. These observations have led to someconcerns over the “variation” in the safety of iPSCs [89]. But claims ofgenerating “transformation-deficient” [45] and thus “safe-inducedpluripotent stem cells (safe-iPSCs) with therapeutic potential” [46] arestill being made. It has been believed that “although there are commonpathways activated during reprogramming and tumorigenesis, pluripotentstem cells and tumorigenic cells have important differences” and thus“the critical distinctions between true cancer cells and reprogrammedsomatic cells may be that reprogrammed cells remain genetically intact”[61]. Thus, despite a clear message arguing the intrinsic distinctionsbetween iPSCs and ESCs [90-92] and some later experimental reportssupporting this argument [93-94], leading iPS researchers still rejectthe intrinsic distinctions between iPSCs and ESCs and the highsimilarity between iPSCs and cancer cells [95]. The most recent reviewon iPSCs still claims: “Numerous studies indicate that, at least forsome clones, iPSCs are similar if not indistinguishable from ESCsderived from embryo or nuclear transfer experiments” and “somatic cellscan be reprogrammed to a pluripotent state, which is molecularly andbiologically indistinguishable from that of ESCs” [28] and a straighttalk states that there's no reason . . . to think that true bona fideiPSCs cannot function as well as ESCs [47]. This attitude is alsoreflected by the lack of appreciation of the cancerous nature of iPSCseven by the well experienced stem cell researchers.

Amazingly, even some cancer researchers apparently still lack anunderstanding of the cancerous nature of the iPSCs. A recent study hasfound “a Myc network accounts for similarities between embryonic stemand cancer cell transcription programs” [96]. Even though some iPSCswere also included in this study, the report failed in identifying iPSCsas cancer cells. As a matter of fact, the corresponding author of thisreport even did not answer the straight question from the presentinventor on whether or not iPSCs are cancer cells.

However, it is hoped that, continued dissection of the iPS reprogrammingprocess may lead to not only a comprehensive identification of asufficient factor set for complete and safe somatic to pluripotentreprogramming [97] but also a increased awareness of the neoplasticnature of iPS reprogramming [39, 98]. More importantly, if theapplication of this invention is placed into practice, the cancerousnature of iPSCs may be made very obvious, if anti-neoplastic agentsdiscovered via methods disclosed in this invention are also veryeffective in killing natural cancer cells.

It is important to point out that iPS reprogramming can turn normalcells neoplastic even without any genetic modification and a cellreproduction event. This feature will be a key point for the presentinvention which is focused on discovering anti-neoplastic agents thatare effective in inhibiting the metabolic mutation serving as a rootprocess for supporting the malicious competition of neoplastic cellsagainst normal cells.

In the past, cancer research has been heavily focused on geneticmutations, including mutations in mitochondria, as causes for neoplasia[99-100]. The discovery of Warburg effect even led some researchers tobelieve that neoplastic cells have abnormal mitochondria. The outcome ofthis genetic cancer dogma is the focus of searching anti-cancer drugsthat fix the genetic mutations, including mitochondria mutations [101].However, many drugs targeting the effects of genetic mutations oftenfail in killing tumor cells and even succeed in killing normal cells[102].

It turns out that, many times, it is the mitochondrial uncoupling, theabrogation of ATP synthesis by mitochondria, promotes the Warburg effectin some neoplastic cells and contributes to their resistance tochemotherapy targeting mitochondria [103]. These cancer cells may shiftto the oxidation of non-glucose carbon sources to maintain mitochondrialintegrity and function [103]. More importantly, increased level of c-Mycin cancer cells causes an increase in level of glutaminase, a proteinthat helps cells convert amino acid glutamine into an energy source. Thebreakdown of glutamine provides cancer cells a carbon source. In fact,glutamine can serve as a major nutrient for cancer cells [104],especially when facing glucose deprivation [105]. Also worth of noticeis that mutation in some genes such as KRAS or BRAF often lead toup-regulation of the expression of GLUT1 (encoding glucosetransporter-1) and SGLT1 [106]. Thus neoplastic cells often haveenhanced glucose uptake and glycolysis, and can survive even at lowglucose concentration [107]. Thus, neoplastic cells may still havenormal mitochondria despite their abnormal use. Amazingly, drugstargeting mitochondria sometimes kill normal cells more effectively butexert less or even no harm to neoplastic cells which use less or evenshut down their mitochondria.

A metabolic mutation or a metabolism switch may be a predominant featurein cancer cell formation. This change may happen at the epigeneticlevels. A simple RNA splicing which is a modification of an RNAtranscript through removing of introns and joining of exons may producedifferent proteins out of the same gene [108-109]. This epigeneticregulation plays a very important role in normal development as well asneoplastic tumorigenesis [110-111]. Very often, the alternative splicingchanges the mode of mass/energy metabolism [112-113] and this alterationin splicing can be influenced by the conditions in which the cellsreside [114-115]. A very recent study just confirmed that somecancer-related epigenome changes have been found in iPSCs [87].

Studies have shown that oncogenes such as Myc [116-117] play veryimportant roles in contributing to glycolytic metabolism in cancer cells[70]. Studies also show that glucose deprivation induces oncogenicmutations [107]. The fact that hypoxia enhances the generation of iPSCs[118] indicates that iPSCs may have switched into a neoplasticglycolysis.

Thus, it is reasonable to expect that iPSCs can be reliably used as aclass of CROFs for screening anti-neoplastic agents that are effectivein inhibiting metabolic mutation which serves as a root for neoplastictransformation of normal cells into tumor/cancer cells.

Of course, this proposal is apparently against the current mainstreamthinking which regards iPSCs as ethical replacements for ESCs and“cancer-free” and thus even “safe” for cell therapy and regenerativemedicine. However, it is right from those reports making “cancer-free”claims for iPSCs that the inventor of this patent application foundevidence of the cancer risk for iPSCs [48]. With more detailed reasoningdisclosed here for linking iPS reprogramming with neoplastictransformation, iPS researchers should come to a reality that theiriPSCs may find a better utility: serving as serendipitous cancer cellsfor screening anti-neoplastic agents.

According to the present invention, potential therapeutic agents may bescreened for their ability for inhibiting aerobic glycolysis inneoplastic cells. Aerobic glycolysis has been studied for many decadesnow and the technical practitioners in the related research field shouldbe familiar with the arts of how to examine the effect of various agentson the different aspects of aerobic glycolysis. Studies can be alsodesigned to study the specific gene expression and/or the enzymeactivity related to an aspect of aerobic glycolysis. There have beenmany publications reporting such studies and thus methodologies can beeasily obtained and followed. But the Seahorse Bioscience approach(http://seahorsebio.com) may provide a very convenient way to achievethese goals. The approach comprises simultaneous measurement of oxygenconsumption rate (OCR) and extracellular acidification rate (ECAR) inthe presence or absence of the testing compounds and thus determinationof the rate of oxidative phosphorylation (OXPHOS) and glycolysis,respectively. A change from OXPHOS to glycolysis is usually the earliestchange in the process of oncogenic transformation. On the contrary, aninhibition of glycolysis can be used as an early indicator for theanti-neoplastic potential of the testing agent.

According to the present invention, potential therapeutic agents mayalso be screened for their ability in reducing the competitiveness ofneoplastic cells in neoplastic anabolism. Neoplastic anabolism includestwo major aspects: the competitive strength of neoplastic cells ingrabbing and utilizing resources for cell mass production and theexistence of a pathogenic Cori cycle that provides resources toneoplasm. Any means known in the art to measure reduction in thecompetitiveness of neoplastic cells and blockage of the pathogenic Coricycle can be used for checking the effect of the screened agents inaffecting these processes in CROFs such as iPSCs. For example, studiescan be designed to measure the gene expression or the enzyme activityrelated to an aspect of neoplastic anabolism.

According to the present invention, potential therapeutic agents may bescreened for their ability of inhibiting invasion, migration andmetastasis of neoplastic cells. This kind of studies has been routinelyperformed with other cancer cells and the methods available in publishedliterature can be easily found and followed.

According to the present invention, potential therapeutic agents mayalso be screened for their ability for inducing death of neoplasticcells. There are many methods for assessing cell viability and some ofthem are routinely used and thus well known.

It should be pointed out that, despite the nonobvious nature of thepresent invention, the enablement of this novel invention is notdifficult at all. Prior arts in studying effects of agents on cancercell metabolism, growth, reproduction, invention, and migration areabundant and very easy to find. Nevertheless, some essential enablementdescriptions of the present invention are still presented here so thatresearchers with ordinary skills in the field can carry out theinvention without undue experimentation.

One embodiment of carrying out the present invention of screeninganti-neoplastic agents using CROFs including iPSCs comprises thefollowing steps:

First, the metabolic status of CROFs such as iPSCs should be evaluatedso that their suitability for screening different kinds ofanti-neoplastic agents can be assessed and a background control can beestablished. This metabolism diagnosis can be done with a Seahorse XFextracellular flux analyzer and there are rich information on SeahorseBioscience website(http://seahorsebio.com/products/xf-analyzers/index.php) forunderstanding the principles underlying the assays and learning how toperform the various tests. Successful applications revealed in scholarpublications can also be found there. If the analysis shows apredominance of glycolysis in the tested cells, then not only the natureof testing cells as bona fide neoplastic cells is confirmed, but alsothe suitability for working as a model for screening anti-glycolysisbroad spectrum anti-neoplastic agents is established.

Secondly, the testing agents will be evaluated on their effects on themetabolism of the CROFs, such as iPSCs. Again, this evaluation can beperformed using the Seahorse XF extracellular flux analyzer. If thetesting agents can inhibit the glycolysis more than other aspect of themetabolism, then the chance of them serving as effective metabolicroot-level anti-neoplastic agents is great and their side-effect may beminimal.

Thirdly, the metabolic effective testing agents can be evaluated furtherfor their capability to kill the testing cells. It is well known in theart that viability of a cell can be determined by contacting the cellwith a dye and viewing it under a microscope. The most common dye usedin the art for this purpose is trypan blue. Viability of cells can alsobe determined by detecting DNA synthesis. Cells can be cultured in cellmedium with labeled nucleotides, e.g., ³H thymidine. The uptake orincorporation of the labeled nucleotides indicates DNA synthesis. Inaddition, colonies formed by cells cultured in medium indicate cellgrowth and is another way to test viability of the cells.

Fourthly and as a particularly suited approach to reduce to practice thepresent invention, novel anti-neoplastic agent(s) could be testedagainst CROFs in tetrazolium salt-based metabolic assays such as the XTTassay. As such, the respective CROFs that are maintained and propagatedin cell culture medium (e.g. RPMI 1640/10% FCS) could be plated in96-well plates and then incubated for a given time period (e.g. 48 to 72hours) with the respective testing agent(s), using no testing agent(s)group as a control. At the end of such incubation, XTT substance (andits activation reagent) would be added and the level of soluble formazanproduct derived from XTT by cellular enzymes would be measured. The morecell proliferation coinciding with metabolic activity is present, themore formazan would be produced by the cells from XTT. If, however,there is an active anti-neoplastic agent equally present, such formazanproduction would be significantly reduced as a result of thegrowth-inhibitory effect of such compound.

All of these mentioned and many other unmentioned cellviability/proliferation tests are well-established and thus do not needany more detailed description here.

Fifthly, the metabolic effective testing agents can be evaluated furtherfor their capability to inhibit the invasion and migration of CROFsincluding iPSCs. These evaluations can be performed with arts known inthe field of cancer research. For example, the invasion and migrationcapability can be studied using a microfluidic device [119]. The effecton metastasis can be evaluated by established methods.

As a sixth point, the validity of the various anti-neoplastic effects ofthe selected anti-plastic agents from testing with CROFs such as iPSCsmay be confirmed with natural cancer cells that are well characterized.Although this additional test is not an intrinsic component of thepresent invention it is nevertheless helpful for establishing thepresent invention as a trustable and reliable method for screeninganti-neoplastic agents.

Finally, CROFs could be injected in vivo into nude mice, subsequentlyobserved as to whether they grow out to develop macroscopic tumors, and,if so, treated by means of novel anti-neoplastic drug candidates withprimary tumor shrinkage as one of the possible endpoints for drugefficacy.

Further than providing the above guidelines and continuing onilluminating the likely neoplastic processes in CROFs such as iPSCs andthus showcase some potential applications of using CROFs including iPSCsfor exploring a variety of anti-neoplastic agents, some additionalexamples of recent discoveries on metabolic mutation-based neoplastictransformation are presented below. Methods of discoveringanti-neoplastic agents described in these reports may also serve asillustrations of additional embodiments of the current invention.

It has been shown that hypoxia and oncogenic mutations drive glycolysis,with the pyruvate to lactate conversion being promoted by increasedexpression of lactate dehydrogenase A (LDH-A) and inactivation ofpyruvate dehydrogenase. The NAD+ pool is consecutively regenerated andsupports the high glycolytic flux required to produce anabolicintermediates. Glutaminolysis provides metabolic intermediates such asalpha-ketoglutarate to feed and thereby maintain the tricarboxylic acidcycle as a biosynthetic hub. Glycolysis and glutaminolysis share thecapacity to generate NADPH, from the pentose phosphate pathway andthrough the malate conversion into pyruvate, respectively. Both pathwaysultimately lead to the secretion of lactate. More than a waste product,lactate was recently identified as a major energy fuel in tumors.Lactate produced by hypoxic tumor cells may indeed diffuse and be takenup by oxygenated tumor cells. Preferential utilization of lactate foroxidative metabolism spares glucose which may in turn reach hypoxictumor cells. Monocarboxylate transporter 1 regulates the entry oflactate into oxidative tumor cells. Its inhibition favors the switchfrom lactate-fuelled respiration to glycolysis and consecutively killshypoxic tumor cells from glucose starvation. Combination withradiotherapy renders remaining cells more sensitive to irradiation,emphasizing how interference with tumor cell metabolism may complementcurrent anticancer modalities [120]. On the other hand, increasedexpression and activity of LDH-A were detected in Taxol-resistant cellswhich showed a higher sensitivity to the specific LDH inhibitor,oxamate. Treating Taxol-resistant cells with the combination of Taxoland oxamate showed a synergistical inhibitory effect on these cancercells by promoting apoptosis in these cells [121].

Neoplastic cells often possess different capacities than normal cells indealing with some metabolite [106]. To eliminate lactate and to preventcellular acidification tumor cells show up-regulation of MCT4, anH+-coupled lactate transporter. In addition, the Na+-coupled lactatetransporter SMCT1 is silenced in cancer cells. SMCT1 also transportsbutyrate and pyruvate, which are inhibitors of histone deacetylases. Thesilencing of SMCT1 occurs in cancers of a variety of tissues.Re-expression of SMCT1 in cancer cell lines leads to growth arrest andapoptosis in the presence of butyrate or pyruvate, suggesting that thetransporter may function as a tumor suppressor. Tumor cells meet theiramino acid demands by inducing xCT/4F2hc, LAT1/4F2hc, ASCT2, and ATB0,+.xCT/4F2hc is related primarily to glutathione status, protection againstoxidative stress, and cell cycle progression, whereas the other threetransporters are related to amino acid nutrition. Pharmacologic blockadeof LAT1/4F2hc, xCT/4F2hc, or ATB0,+ leads to inhibition of cancer cellgrowth.

An epigenetic mechanism of the Warburg effect has been proposed [122].Fructose-1,6-bisphosphatase-1 (FBP1), which functions to antagonizeglycolysis was down-regulated through NF-kappaB pathway inRas-transformed NIH3T3 cells. Restoration of FBP1 expression suppressedanchorage-independent growth, indicating the relevance of FBP1down-regulation in carcinogenesis. Indeed, FBP1 was down-regulated ingastric carcinomas and gastric cancer cell lines. Restoration of FBP1expression reduced growth and glycolysis in gastric cancer cells.Moreover, FBP1 down-regulation was reversed by pharmacologicaldemethylation. Its promoter was hypermethylated in gastric cancer celllines and gastric carcinomas. Inhibition of NF-kappaB restored FBP1expression, partially through demethylation of FBP1 promoter.

Using iPSCs as a group of CROFs for screening anti-neoplasticagents/drugs has many advantages which include but are not limited to:

First, there are many different types of iPSCs which are readilyavailable for research use;

Secondly, many iPSCs have been well characterized in their geneticaspects and even some epigenetic aspects and thus further exploration ontheir metabolic mutation-based neoplastic changes would be moreproductive than testing on other less known cells.

Thirdly, iPSCs have become a part of some researchers' scientific life.These dedicated iPS researchers seating on the iPS bandwagon wouldbecome valuable human resource in carrying out the present invention ofusing iPSCs as target cells for screening anti-neoplastic agents, if thefinancial incentive is given for killing (neoplastic) iPSCs rather thancreating (therapeutic) iPSCs.

Therefore, once iPS researchers realize that enhancing the efficiency ofgenerating iPSCs is not a boosting of the immortalization but actuallyan intensification of the neoplastic transformation, then these iPSresearchers may be the first followers in chasing the discovery ofcancerous iPSCs and become the main force using established iPSCs assurrogates for natural cancer cells to search for broad-cancer-spectrumanti-neoplastic agents. This trend is not obvious at the present time.But with the disclosure of this patent application to the public in thefuture, this trend will for sure to come.

Thus, with an totally unexpected and even unwelcomed discovery of iPSCsas man-made cancer cells and with a detailed presentation linking iPSreprogramming with neoplastic transformation, it is anticipated thatfuture research on neoplasia, which include all kinds of tumors andcancers, will move into a new horizon. With the finding of anti-cancerdrugs that are toxic only to neoplastic but not normal cells, clinicaltreatment of neoplastic diseases may yield an unprecedented goodoutcome.

It should be pointed out that the above guidelines and some examplesrepresent just some possible applications and embodiments of the presentinvention. It should not be understood as limitation and boundary of thepresent invention. The real scope and the right of intellectual propertyprotection should be based on the claims granted for this patentapplication.

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1. A method of screening anti-neoplastic agents by using cellsreprogrammed with oncogenic factors (CROFs), comprising the steps of:determining neoplastic capabilities of CROFs in the presence and in theabsence of a test agent; selecting a test agent which causes asignificant reduction in the neoplastic capabilities of the CROFs. 2.The method of claim 1 wherein CROFs are oncogenic cells produced throughmolecular reprogramming.
 3. The method of claim 1 wherein CROFs includeincorrectly programmed stem cells.
 4. The method of claim 1 wherein theneoplastic capabilities include one or several of the following aspects:elevated aerobic glycolysis, neoplastic anabolism, fast reproduction(proliferation), aggressive invasion or dispersive migration(metastasis).
 5. The method of claim 1 wherein the reduction ofneoplastic capabilities is judged by comparing a treatment group (in thepresence of a single test agent or a combination of testing agents) witha control group (in the absence of such agent(s)) of the same type ofCROFs.
 6. The method of claim 3 wherein the incorrectly programmed stemcells include induced pluripotent stem cells (iPSCs) which are somaticcells induced with one or more of transcription factors commonly knownfor generating iPSCs.
 7. The method of claim 5 wherein the reduction ofneoplastic capabilities can be revealed by examining aerobic glycolysisfor an indication includes decreased extracellular acidification rate(ECAR) or decreased expression of glycolytic-specific enzymes.
 8. Themethod of claim 5 wherein the reduction of neoplastic capabilities canbe revealed by examining neoplastic anabolism for an indication includesreduced rate of glutaminolysis or reduced expression of glutaminolyticenzymes.
 9. The method of claim 5 wherein the reduction of neoplasticcapabilities can be revealed by examining cell viability or reproductionrate.
 10. The method of claim 5 wherein the reduction of neoplasticcapabilities can be revealed by examining cell invasion, migration ormetastasis.